Many viruses synthesize mRNAs containing an internal ribosomal entry site (IRES) that mediates cap-independent translation. IRESs differ greatly from one another and use distinct mechanisms for initiation. Our studies of the molecular mechanisms of initiation on 4 different IRESs will provide a basis for understanding their cell type-specificity, for the design of inhibitors, and may identify underlying mechanistic similarities. IRESs often act in a cell type-specific manner that determines viral pathogenesis. The attenuated neurovirulence of poliovirus vaccine strains is due to defective IRES function, likely due to a need for cell-specific IRES trans-acting factors (ITAFs). We will identify the complete set of canonical factors/ITAFs required by this IRES using biochemical reconstitution of initiation in vitro, determine how ribosomes reach the initiation codon approximately 60 nucleotides downstream of the IRES and map the interactions of components of the translation apparatus with IRESs of attenuated and virulent PV strains. This analysis will elucidate an important aspect of viral tissue tropism. Hepatitis A virus contains a 580 nucleotide-long IRES that also determines viral growth characteristics and pathogenicity in humans. We shall use similar approaches to determine the complete set of canonical factors and ITAFs needed by this IRES and to map their interactions with the IRES. Hepatitis C virus (HCV) and Cricket paralysis virus (CrPV) IRESs use very different mechanisms of initiation. The HCV IRES binds both eIF3 and the 40S ribosomal subunit and then needs only eIF2/GTP/tRNA to form a 48S complex. We shall characterize interactions that lead to 48S complex formation in detail, examine regulation of eIF3's activity during chronic HCV infection and determine how ribosomal subunits join on the IRES. We shall also identify how and where the CrPV IRES binds the 40S subunit and whether its binding excludes eIF2/tRNA from the P site. We shall determine how This IRES induces translocation of aa-tRNA from ribosomal A to P site without concomitant peptide formation so that protein synthesis can begin. We shall reconstitute this process in vitro to determine how this occurs.